The present invention relates generally to diagnosis of filarial nematode infection and, more specifically, to improvements in immunological methods for quantitative detection of adult stage Dirofilaria immitis-associated antibodies in biological fluids.
Filarial nematode infections of humans constitute a worldwide problem estimated to adversely affect the lives of 300 million people. The effects of filarial infection on domesticated animals are correspondingly immense. In the United States alone, millions of dollars are spent each year on treatment of infection in cats, dogs and other pets and agricultural animals. Contributing to the magnitude of the filarial problem is the fact that accurate diagnosis by common diagnostic means remains exceedingly difficult. The absence of reliable procedures for diagnosis of filarial infection has retarded research and development in curative and prophylactic therapy.
Dirofilaria immitis is a filarial nematode parasite which, in its adult form, commonly infects the right ventricle, pulmonary artery and adjacent pulmonary vasculature, frequently causing serious tissue damage and death. Infection of dogs with this particular filarial nematode (commonly referred to as "heartworm") is prevalent throughout the world. Patent, or mature D. immitis infection in dogs can rather readily be treated through administration of drugs such as thiacetarsamide. Drug therapy is often withheld pending certain diagnosis of D. immitis infection, however, because such drugs can be highly toxic to the canine host and the cost of therapy is often prohibitive. Further, the presence or absence of filarial infection may affect the course of treatment of other disease states. As one example, certain drugs useful in treatment of canine intestinal parasites should be avoided because they may be lethal to heartworm, causing fatal embolisms in the recipient animal.
Diagnosis of D. immitis infection can be made with certainty where characteristic microfilariae (thread-like D. immitis embryonic forms) can be visually detected through examination of body fluids. The presence of detectable numbers of microfilariae indicates that infection has progressed to a state of worm maturity sufficient to cause substantial and frequently irreversible tissue pathology.
Parasitological diagnoses are falsely negative when an infected dog displays a so-called "amicrofilaremic" state. Visual detection of microfilariae will not be possible in early stages of infection in which none or too few of the embryonic organisms are circulating. In the case of single sex infections and in old infections wherein the female worm is no longer reproductively capable, no microfilariae will be formed. Further, an amicrofilaremic state may exist as a result of the host's own protective immune response, which is directed to the microfilarial parasite stage only. Specific antibodies clear the embryos from the host's circulation, leaving the adult worms alive.
Numerous serological procedures have been proposed for detection of filarial infection, but none has been widely accepted as a reliable alternative to parasitological diagnosis. As one example, an assertedly highly specific and sensitive procedure for detection of specific, anti-microfilarial antibodies has been proposed by, e.g., Wong, et al., Am. J. Vet. Res., 40, pp. 414-420 (1979). Briefly put, the test employs intact microfilariae in an indirect fluorescent antibody (IFA) procedure to detect circulating anti-microfilarial antibodies. The procedure thus has an advantage in utility for detecting filarial infection wherein an amicrofilaremic state exists as a result of the infected animal's protective immune response to the presence of the microfilariae. Apart from the fact that the procedure requires expensive fluorometric apparatus generally unavailable in veterinary practice, the test is not effective in diagnosing disease states which are amicrofilaremic as a result of, e.g., single sex infections, and falsely negative results may be nearly as common as in the parasitological procedures. Even when successful, the procedure shares with parasitological detection the disadvantage of ascertaining only relatively advanced disease states.
It is known that the presence of adult nematodes in an infected host does produce a humoral immune response and it has been suggested that reliable assays directed toward detection of adult D. immitis-associated antibodies would be exceptionally useful in providing information upon which a therapeutic or prophylactic treatment regimen can be selected.
Proposed enzyme-linked immunosorbent (ELISA) serological assays based on detection of antibodies associated with adult stages of worms have been lacking in reliability and ease of performance. Contributing to the lack of specificity of such procedures is the polyantigenicity of the parasite and the consequently varied immunological response of the host animal. It is widely held, for example, that filarial nematode infection presents the host with an array of antigenic stimuli of varying intensity and duration. Discrete "antigens" which may provoke development of correspondingly discrete circulating antibodies have been associated not only with various body parts of the adult nematode (e.g., the "cuticular" and "cytoplasmic" antigens) but also with nematode growth products ("metabolic" antigens) and, of course, differing growth stages (e.g., the microfilarial-associated antigens).
Crude and semi-purified soluble antigens (like the above-noted "microfilarial" antigens) can be obtained by simple extraction of whole live adult worms and these have been found to be effective in detecting specific adult D. immitis-associated antibodies in ELISA assays. Such antigen preparations, however, appear to be additionally cross-reactive with unspecified antibodies present in many serum specimens of both infected and uninfected dogs. Procedures based on the use of adult D. immitis-associated antigens have therefore provided falsely positive results.
There exists, therefore, a substantial need to improve the specificity of immunological serodiagnostic assays for adult D. immitis-associated antibodies and to reduce the complexity of the procedures so that they can be readily performed in routine veterinary and clinical practice. Such improvements in the serodiagnosis of D. immitis infection would aid in the early, accurate detection of infections in which the characteristic microfilariae are circulating in the body fluids and in established amicrofilaremic infections in dogs.
Specifically incorporated by reference herein for the purposes of indicating the background of the invention and illustrating the state of the art are the following publications of the inventor and his co-workers: Grieve, et al., Am. J. Vet. Res., 42, pp. 66-69 (1981); and Grieve, et al., Int. J. Parasitol 9, pp. 275-279 (1979).